Infected cells containing hyper-replicating cytosolic S. Infantis did not undergo apoptosis. (A) Detection of intracellular bacterial load using the gentamicin protection assay, without (total intracellular bacteria, red curve) or with (cytosolic bacteria, blue curve) chloroquine. Data were obtained from three independent replicates of each sample at 0, 2, 4, 6, 8, 10, and 12 hpi. (B) Detection of the distribution and proportion of two subpopulations in Caco-2 cells by immunofluorescence staining. SI, S. Infantis. Red: Salmonella. Green: Lamp-1. Blue: DAPI. Scale bar, 10 µm. (C) Cell viability assay of Caco-2 cells infected with S. Infantis within 12 h. CN, control. (D) Western blot analysis of Caspase-9, Cleaved-caspase-3, and Cleaved-PARP protein expression levels within 12 h after bacterial infection. A total of 10 time points were set for sampling: 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 hpi. CN, control. ns, no significant difference. (E) Immunofluorescence staining analysis of Cleaved-caspase-3 in Caco-2 cells. Samples were treated at 1, 4, and 8 hpi, respectively. CN, control; SI, S. Infantis. Red: S. Infantis. Green: Cleaved-caspase-3. Blue: DAPI. Scale bar, 10 µm. CCCP was the apoptosis-positive control group. Data were presented as the mean ± SEM from three independent experiments (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.